The long-term objective of this project is to understand how a eukaryotic chromosome replicates. To this end, a replication map of chromosome III of the yeast, Saccharomyces cerevisiae, has been prepared. This replication map includes information about the positions of replication origins, determined by 2D gel analysis of replication intermediates, the positions of ARS elements, detected by their ability to promote autonomous maintenance of plasmids, the positions of replication termination. This proposal addresses three major issues concerning replication origins in this chromosome and the cis-acting replicators necessary for their function. First, the structure of several replicators will be further defined using in vitro mutagenesis to create mutant ARS elements whose function will be in plasmids and in their normal chromosomal context. These mutant replicators will also be used in genetic screens and biochemical studies to identify proteins that interact with replicators. Of particular interest will be the identification of proteins other than origin recognition complex and ARS binding factor 1 that interact with replicators in vivo. Second, the mechanism(s) by which chromosomal replicator activity is influenced by chromosomal context will be studied by determining the effects on replicator activity of mutations in genes known to be involved in position effects on transcriptional activity and by mapping the elements required for context effects on replicator function. Finally, a newly discovered homology-dependent mechanism for the maintenance of chromosomes lacking functional replicators will be studied be determining the requirement for known genes involved in recombination and cell cycle checkpoints in the process, screening for mutants defective in the process, and by studying the chromosome topology dependence of the process. These studies should greatly increase our understanding of a fundamental cellular process, chromosomal replication. These questions cannot be readily approaches in larger eukaryotes at this time because cis-acting replicator sequences have not been identified and characterized, and because the extremely large size of their chromosomal DNA's make isolation and manipulation difficult. Knowledge gained from the yeast system should be applicable to larger eukaryotes, and may lead to an understanding of chromosomal rearrangements the reactivation of DNA replication that are characteristic of malignant cells.